1. Field of the Invention
The present invention relates generally to the fields of chemistry, molecular biology, and diagnostics. More particularly, it concerns methods and compositions for obtaining a high affinity synthetic capture agent for a molecular or biomolecular target by co-immobilizing at least two low-to-moderate affinity ligands on a suitable surface. Such a high affinity synthetic capture agent is referred to as a mixed element capture agent (MECA). MECAs will be of great utility in the construction of medical diagnostic devices.
2. Description of Related Art
There is great interest in the development of techniques with which to monitor the levels, post-translational modification states and activities of large numbers of proteins simultaneously. One approach is to construct protein-detecting arrays (Kodadek, 2001; Kodadek, 2002), akin to the DNA microarrays used widely in genomics research. Such devices may be comprised of many different protein-binding molecules arrayed on a suitable surface in a defined pattern, each capable of recognizing its target protein with high affinity and high specificity. These high affinity and high specificity protein-binding molecules or ligands are referred to as capture agents. An alternative format is to immobilize protein-binding molecules on encoded beads (Vignali, 2000; Oliver et al., 1998). A significant challenge in the development of such technology is the isolation of large numbers of protein-binding compounds with a sufficiently high binding affinity and specificity to be useful in the capture of particular proteins from a complex mixture.
Most of the effort in this area has focused on the use of macromolecular biomolecules as capture agents, such as antibodies (Eggers et al., 1998; Hunag et al., 2002; Huang, 2001; Wiese et al., 2001; Huang et al., 2001; Walter et al., 2000; and Haab et al., 2001), nucleic acids (particularly RNA) aptamers (Osborne et al., 1997; Jhaveri et al., 2000; Famlouk and Jenne, 1998; Seethsnunan et al., 2001; and Vaish et al., 2002) and protein-RNA fusions (Roberts and Szostak, 1997; Wilson et al., 2001; and Colas et al., 1996).
Protein-binding molecules can be readily isolated from combinatorial libraries or other types of large compound collections using a number of methods. Unfortunately, small molecules, peptides, peptidomimetics and other synthetically accessible compounds rarely bind to their target protein with an affinity comparable to that of a good antibody (equilibrium dissociation constant (KD)≦10−9 M). Instead, small molecule/protein complexes generally exhibit KDs in the μM range, with the exception of molecules optimized through extensive medicinal chemistry efforts or natural selection. This modest affinity is insufficient to capture low abundance proteins from complex mixtures. In addition, the relatively rapid dissociation rates of such complexes result in the loss of most of the bound capture target during the inevitable washing steps required to minimize non-specific “background” binding of high abundance or “sticky” proteins. Therefore, a central problem in applying organic chemistry to the development of protein-detecting microarrays is obtaining higher affinity synthetic ligands in a high-throughput fashion.
In the case of pharmaceuticals, optimization of a lead compound is generally achieved through a tedious and labor-intensive process in which hundreds of relatives of the lead molecule are synthesized and evaluated for activity. It is not possible to apply this approach on a scale where ligands are required for hundreds or even thousands of proteins. One potential “shortcut” in the path from low to high affinity agents is to employ multivalency. For example, coupling two or more modest affinity protein ligands with an appropriate linker can provide a high affinity bivalent or multivalent ligand (Shukery et al., 1996; Olejniczak et al., 1997; Thorn et al., 2001; Terskikh et al., 1997; Merritt et al., 2002; Kitov et al., 2000; Kiessling et al., 2000 and Cussac et al., 1999). Unfortunately, linker optimization can be time consuming, and most approaches to this problem are unsuitable for high-throughput proteomics applications (see Maly et al., 2000, for an exemplary combinatorial approach). Thus, there remains a need for rapid, selective and high-affinity compositions and methods for rapidly providing a high affinity synthetic capture agent.